BHT Analysis in Polyethylene Film

J. Dairy Sci. 91:11–19doi:10.3168/jds.2007-0464© American Dairy Science Association, 2008.

Release of Butylated Hydroxytoluene from an Active Film Packagingto Asadero Cheese and Its Effect on Oxidation and Odor Stability

C. D. Soto-Cantu, A. Z. Graciano-Verdugo, E. Peralta, A. R. Islas-Rubio, A. Gonzalez-Cordova,A. Gonzalez-Leon, and H. Soto-Valdez1

Centro de Investigacion en Alimentacion y Desarrollo, A.C., CTAOV, Apdo. Postal 1735, Hermosillo, Son., 83000 Mexico

ABSTRACT

Antioxidant active packaging consisting of coex-truded films made of low density polyethylene (LDPE)added with 0, 8, and 14 mg/g of butylated hydroxytolu-ene (BHT) and polyamide 6/66 were fabricated. Therelease of BHT from the films to Asadero cheese wasdetermined. Most of the BHT was diffused from theLDPE layer to the cheese during the first 20 d of storageat 5°C. Diffusion coefficient for the diffusion of BHTfrom the films 8 and 14 to the cheese was calculatedas 6.24E-12 and 6.26E-12 cm2/s, respectively. The re-lease of BHT from the film added with 8 mg/g of theantioxidant in the LDPE layer complied with the legallimit established for food products. However, the filmadded with 14 mg/g of the antioxidant exceeded thatlimit. The film added with 8 mg/g of BHT maintainedthe same levels of oxidized odor from 20 to 100 d ofstorage.Key words: antioxidant packaging, butylated hydroxy-toluene, migration, Asadero cheese

INTRODUCTION

Milk fat contains approximately 2.3% palmitoleicacid (C16:1n-7), 24.6% oleic acid (C18:1n-9), and 2.8%linoleic acid (C18:2n-6) with the last being susceptibleto lipid oxidation (van Aardt et al., 2005). Asadero is awhite and semihard cheese of Mexican origin with ashelf life of 60 d at refrigerated storage. The manufac-turing process includes the use of high temperatures(75 to 80°C) to melt the curd, to shape it with a mold,or to stretch and roll it in different shapes. During thisstage the fat is concentrated and the heat applied mayprovoke the initiation of lipid oxidation reactions. Asad-ero cheese is commercially packed under vacuum withtransparent barrier films. Most cheeses are packed intransparent materials and exposed to light during retail

Received June 20, 2007.Accepted September 28, 2007.1Corresponding author: [email protected]

11

storage increasing the susceptibility for light-inducedoxidation with the development of off-flavors. Volatilecompounds like hexanal, heptanal, octanal, nonanal,2-butanol, 2-pentanol, acetaldehyde, benzaldehyde, 2-pentyl-furan, and tetrahydrofuran (THF) have beenidentified as being responsible for these unpleasant off-flavors in packaged semihard cheeses (Urbach, 1993;Mortensen et al., 2002; Sunesen et al., 2002; Juric etal., 2003; Holm et al., 2006). The production of thesevolatile compounds is decreased by adding antioxidants(van Aardt et al., 2005).

Butylated hydroxytoluene (BHT) or 3,5-di-tert-butyl-4-hydroxytoluene is a synthetic antioxidant commonlyused in the food industry. The BHT is lipid solubleand terminates free-radical chain reactions by donatinghydrogen atoms to free radicals producing more stablecompounds. The legal limit for the addition of BHT tomost foods is 200 mg/kg of fat (Code of Federal Regula-tions, 2007a). Usage of active packaging in contact withfood is an alternative for adding antioxidants to main-tain a constant concentration of the additive in the foodduring storage instead of adding high levels of additivesdirectly. This beneficial role of antioxidants in packag-ing films has led to research in the manufacture ofantioxidant-added films and their utilization in variousfood-packaging situations (Chick, 2002). Addition of an-tioxidants to polyolefins is a common practice duringfilm manufacture because they are also used to protectlow density polyethylene (LDPE) from degradation.The BHT is usually used at concentrations up to 500mg/kg in polyolefins (Linssen et al., 1998). During filmprocessing, part of the antioxidant is lost because of itsability to function as a free-radical scavenger. Also, itmay be lost to the environment because of its highvolatility at processing temperatures (Vulic et al.,2002). Therefore, to ensure the extension of the shelflife of food susceptible to deterioration by oxidation,active packaging films must be formulated with ahigher antioxidant concentration than common polyo-lefin films. Studies of migration of BHT from monolayerpolyolefin films have shown that BHT has a high mobil-ity toward fatty foods and to the environment (Wessling

SOTO-CANTU ET AL.12

et al., 1998, 2000; Galindo-Arcega, 2004; Torres-Arreolaet al., 2007). Because BHT can be used as an additivefor direct addition to food (Code of Federal Regulations,2007b), its use in active packaging has lead to commer-cial applications, such as in the liners for breakfastcereal packages (Yanidis 1989).

Migration is the result of the diffusion and equilib-rium process involving the transfer of low molecularmass compounds (additives) from a plastic packaginginto a food or food simulant. The migrants diffusethrough the amorphous portion of the polymer matrixtoward the interface where they are partitioned be-tween the 2 media until their chemical potential valuesin both the polymer and the food reach the equilibrium.Migration is often described by Fick’s second law. Thetheoretical background of the migration process is de-scribed by Crank (1975), Hamdani et al. (1997), andGarde et al. (2001). Till et al. (1982) reported diffusioncoefficient (D) values of 3.7E-11 and 4.2E-12 cm2/s formigration of BHT from a high density polyethylene(HDPE) plaque (235-�m thickness and 100 mg of BHT/kg) to corn oil at 40 and 21°C, respectively. Han et al.(2003) reported D values of 2.34E-11, 8.65E-11, and2.20E-10 cm2/s for BHT in a film made of a mixture ofHDPE and LDPE (30.5-�m thickness) to ethanol at 23,31, and 40°C, respectively.

Monolayer LDPE films are not suitable for cheesepackaging due to their high oxygen transmission rates(OTR) that accelerate the oxidation reactions. There-fore, multilayer films made of polyamide, polyethyleneterephthalate, or polyvinylidene chloride are coex-truded or laminated with LDPE to provide a good oxy-gen barrier (Robertson, 2006). These multilayer filmsare widely used not only for cheese packaging but forother food products like chicken breast fillets (Pettersenet al., 2004) and mussel meat (Bindu et al., 2004). Bar-rier properties for different multilayer packaging arereported by Lange and Wyser (2003). In the case ofcheese, the use of oxygen barriers by itself does notassure the absence of lipid oxidation. Oxidation hasbeen reported in Danboo cheese (25% fat) packed inpolyethylene terephthalate trays with an oxygen scav-enger for 84 d at 4°C (Holm et al., 2006). Also, Sunesenet al. (2002) identified 28 volatile compounds in a pro-cessed cream cheese spread (31% fat) packed in glassflasks at 5, 20, and 37°C during 1 yr. Glass is one ofthe best barriers to oxygen.

The present work reports the development of an anti-oxidant active packaging consistent of coextruded filmsmade of polyamide 6/66 (PA6/66) and LDPE added withdifferent concentrations of BHT. The effect of heat pro-cessing on BHT concentration in the films was deter-mined as well as the release and diffusion coefficientof BHT from the coextruded films to Asadero cheese.

Journal of Dairy Science Vol. 91 No. 1, 2008

The effect of the released BHT on cheese oxidation andodor stability was also investigated, and the complianceof the films with the legal limit established for foodproducts was verified.

MATERIALS AND METHODS

Film Manufacture

Three formulations of LDPE (Petrothene, Equistar,Houston, TX) pellets containing 0, 8, and 14 mg/g ofBHT (99% purity, TCI America, Portland, OR) werecoextruded with PA6/66 (Ultramid, BASF, Freeport,TX) by the blow extrusion process with a pilot-plantsize coextruder (Beutelspacher, Mexico DF, Mexico) atthe Centro de Investigacion en Alimentacion y Desa-rrollo, AC campus Hermosillo, Mexico. An ethyleneacrylic acid adhesive (Primacor, Dow, Midland, MI) wasused to adhere the 2 layers. The 0% BHT coextrudedfilm was used as a control. The films were labeled as0, 8, and 14 according to the BHT added to the LDPElayer during their manufacture.

The coextruded films thickness was measured witha micrometer (model DTT, E.J. Cady & Co., Wheeling,IL). The layers thicknesses were measured with a CarlZeiss transmitted light microscope (Axiolab E,Thornwood, NY) equipped with a micrometer.

The OTR of the films was measured using an Oxtran2/20 m (MOCON, Modern Controls Inc., Minneapolis,MN; ASTM D3985-95, 1995). Tests were performed at23°C and expressed as cm3/m2/day. Two replicates weretaken at the thickest and the thinnest section of eachfilm.

Cheese Processing, Packaging, and Storage

Asadero cheese was obtained from one batch of thestandard production from the Rayon cheese factory(Rayon, Sonora, Mexico). This brand of cheese does notcontain any additive. Moisture and fat were determinedaccording to the AOAC methods (AOAC, 2000a,b).Twenty-four blocks of cheese (100 ± 5 g; 7.0 × 10.0 ×1.5 cm) were packed in each of the 0, 8, and 14 filmsfor the BHT release and oxidation experiments. Othersets of 20 blocks of cheese were packed in each of the3 films for the sensory experiment. A vacuum sealerLoma Q NT 300/2 (Doo-Il R.S. Co. Ltd., Kyeongki-Do,South Korea) was used to obtain packages with −1 barof pressure. The packages were stored for 100 d at 5 ±1°C and exposed to fluorescent light (fluorescent lampsplatinum FO32T8; Promolux, Shawnigan Lake, Can-ada) with an intensity ranging between 392 and 1,396lx (luxometer SP-840020, Neurtek Instruments, Gui-puzcoa, Spain). The packages were rotated every 5 d tominimize the differences in light exposition. To have

ANTIOXIDANT PACKAGING FOR CHEESE 13

samples of fresh cheese (from the same batch) availableduring the experiment, enough fresh cheese was frozenat −20°C.

BHT Release Test

Pieces of cheese packed in films 8 and 14 were sam-pled at 0, 3, 5, 20, 60, 80, and 100 d of storage. Thepieces of cheese were unpacked and BHT concentrationwas determined in the coextruded films as explainedbelow. Three replicates were analyzed and results wereexpressed as milligrams per gram of BHT in the films.To calculate the residual BHT in the films, concentra-tions of BHT in samples at time 0 were compared (aspercentage) with the concentration added when thefilms were processed. To obtain the kinetic of release,concentration of BHT in the films was plotted vs. stor-age time. The BHT was not quantified in the cheesebecause once it reaches the product, it may be lost bytrapping free radicals.

Determination of BHT Concentration in theFilms. Pieces of the coextruded films (1 × 1 cm, ∼1 g)were extracted with 250 mL of acetonitrile (JT Baker,Xalostoc, Mexico) with constant stirring under refluxat 60°C for 10 h. A second 10-h extraction was appliedto the same sample to confirm that all the BHT wasextracted in the first extraction. An aliquot of the ex-tract was used for BHT quantification. Three replicateswere carried out for each sample. The BHT quantifica-tion was performed by reverse-phase HPLC using aliquid chromatograph (Varian 9012, Mexico City, Mex-ico) coupled to a fluorescence detector (Varian 9075)with an excitation wavelength of 282 nm and an emis-sion wavelength of 308 nm. A 10-�L sample volumewas injected (Rheodyne 7125 injector) into the HPLCand eluted with methanol:water (98:2, JT Baker) at aflow rate of 1 mL/min for 10 min at 25°C (Wesslinget al., 2000; Galindo-Arcega, 2004). A C18 Omnisphercolumn (150 × 4.6 mm; Varian), protected with a C18guard column (50 mm) was used. A calibration curvefor BHT was prepared from 1 to 50 �g/mL solutions inacetonitrile. Concentration of BHT was expressed asmilligrams per gram of coextruded film. Under thesechromatographic conditions, the retention time for BHTwas 3 min. Recoveries of 83.5 ± 1.7% were determinedby spiking the control film (film 0) with BHT to achievea concentration of 50 mg/kg followed by treatment asabove. All analytical data were corrected for recoveries.The limit of quantitation (LOQ) of the procedure usedin this analysis was determined to be 5.4 mg of BHT/kg in the film.

Determination of D. A simplified migration model(equation 1) reported by Hamdani et al. (1997) for thebeginning of the migration process was used to calculate


D of BHT from the layer of LDPE of the films 8 and 14 tothe cheese. The following assumptions were considered:the BHT diffuses only to the cheese; the BHT concentra-tion in the film decreases during contact (finite packag-ing); the volume of food is much larger than that of thefilm (cheese volume:film volume = 167.4); the contacttime considered is short (20 d).

mF,t

mF,∞=

2L √Dt

π[1]

where mF,t is the mass of BHT diffused to the cheeseafter time t; mF,∞ is the mass of BHT diffused to thecheese at steady state; L is the thickness of the LDPElayer; D is the diffusion coefficient; and t is time. Valuesof mF,t/mF,∞ were plotted vs. t1/2 and D was calculatedfrom the slope (SF,∞), according to equation 2 (Hamdaniet al., 1997).

D =π4 (SF,∞ L)2 [2]

Cheese Oxidation

Preliminary experiments showed that THF was avolatile compound produced by cheese during storageand its production was inhibited by the direct additionof BHT. The THF was determined in cheese samplestaken at 0, 20, 40, 60, 80, and 100 d of storage. Eachcheese sample was homogenized, and a 30-g portionwas introduced to a 40-mL vial fitted with PFTE/whitesilicon septa (Supelco, Toluca, Mexico) and a screw cut-out top cap. Five microliters of internal standard solu-tion [24 mg/mL of 4-methyl 2-pentanone (4M2P) Sigma,St. Louis, MO] in hexane (Sigma-Aldrich, Toluca, Mex-ico) were added before heating at 90°C/30 min. A 25-�L volume of the headspace was taken with a gastightprewarmed syringe and injected in a Varian Star3400CX gas chromatograph equipped with a flame ion-ization detector and a Supelcowax 10 column (25 m ×0.25 mm i.d., film thickness 0.25 �m; Supelco). Theinjector and detector temperatures were 220 and 200°C,respectively. Nitrogen was used as a carrier gas at 1mL/min, and the temperature program started at 30°C/2 min, followed by an increase to 40°C at 1°C/min. Then,an additional increase to 200°C at 10°C/min was ap-plied to clean the system. Three replicates were ana-lyzed, and results were expressed as micrograms pergram of THF in the cheese. A calibration curve for THF(JT Baker) was prepared as follows: vials with 30 g ofhomogenized fresh Asadero cheese were spiked with avolume of 5 �L of standard solutions to achieve concen-trations from 16 to 79 �g/g of THF and a fix concentra-

SOTO-CANTU ET AL.14

tion of 4 �g/g of 4M2P. The mixture was manuallymixed, and the heating process was applied. The ab-sence of THF in the fresh cheese was previously con-firmed. The ratio THF/4M2P areas were plotted vs.THF concentration to obtain a linear graph. Recoveriesof 111 ± 12% were determined by spiking fresh cheesewith known concentrations of THF/4M2P followed bythe heating treatment described above. The LOQ of theprocedure used in this analysis was determined to be12 �g/g of THF in the cheese. Three replicates wereanalyzed for recoveries and LOQ calculations.

Sensory Analysis

Seven people were semitrained in 4 sessions for odoranalysis of the cheese packed in the antioxidant films.Two references of fresh cheese and one of oxidizedcheese (positive sample) were given in 10-g portions.The oxidized cheese was obtained by irradiating acheese sample from the same batch, with UV light for10 min. Two General Electric 15 W G15 T8 lamps wereused (82% of the irradiance emitted in the UV-C, 250to 280 nm, region). The distance between the lampsand the cheese sample was 18 cm. Panelists were askedto sniff each sample and compare it with the positiveone. In a second session, the same samples (nonidenti-fied) were given to the panelists to confirm their abilityto identify the fresh and oxidized samples. Intensitieswere rated on a nonstructured 13 cm scale (0 = nonoxi-dized to 13 = highly oxidized). Five panelists were cho-sen to participate in the sensory analysis. Samples ofcheese packed in the 0 and 8 films and stored at 5 ±1°C were taken at 20, 40, 60, 80, and 100 d. They wereunpacked and given to the panelists in 10-g portionsas in the training sessions. References of fresh (frozenat −20°C) and oxidized cheese (treated as above) werealso given to the panelists in every session to refreshthe nonoxidized and oxidized odor, respectively (Stoneand Sidel, 1985). Two replicates were analyzed everysession. The packed samples were rated on the samescale as above (Pedrero and Pangborn, 1989).

Statistical Analysis

A randomized complete block design with 2 treat-ments (film 0 and 8) in 5 blocks (storage time) wasapplied to the sensory experiment. The ANOVA wasperformed by ANOVA test and significant differencesbetween the means were determined by the Tukey-Kramer test (P < 0.05) using the Number CruncherStatistical System.


Table 1. Thickness and oxygen transmission rate (OTR) of the coex-truded films fabricated of low density polyethylene [LDPE; with butyl-ated hydroxytoluene (BHT)] and polyamide 6/661

Film Thickness2 (�m) OTR3 (cm3/m2 per d)

0 90.97 ± 24.01 24.0 to 47.08 94.95 ± 24.36 21.0 to 38.014 98.42 ± 22.48 26.9 to 41.3

1Thickness of the LDPE layer was 43.55 ± 9.79 �m. Coextrudedfilms fabricated with 0, 8, and 14 mg/g of BHT in the LDPE layer.

2Mean ± standard deviation of 60 replicates.3Two replicates were analyzed for each film, the thickest and the

thinnest section of the films.

RESULTS AND DISCUSSION

Film Characteristics

The thickness of films 0, 8, and 14 ranged between91 and 98 �m (Table 1). Microscopic observationsshowed that the thicknesses of individual layers wereapproximately 43, 3, and 45 �m for the LDPE (BHTadded), adhesive, and PA6/66 layers, respectively. TheOTR values ranged from 21 to 47 cm3/m2/d (Table 1),which are considered adequate for vacuum cheese pack-aging (Robertson, 2006). Moreover, lower OTR valueswill be expected at refrigeration temperature, comparedwith the experimental results at 23°C (Dorado-Rodeloet al., 2007).

The films developed are good oxygen barriers as com-pared with monolayer polymer films, such as LDPE(7,883 cm3/m2/day) for a 17.5-�m film and biorientedpolypropylene (1,642 cm3/m2/day) for a 29.5-�m film(Soto-Valdez and Mendoza-Wilson, 1998). A PA66monolayer film showed an OTR of 51.8 ± 12.9 for a 23.0± 0.5 �m (Dorado-Rodelo et al., 2007). Therefore, theoxygen barrier depends on the polyamide thicknesslayer.

Release of BHT from the Films

BHT Concentration in the Films. The final levelsof BHT in the 8 and 14 coextruded films were 3.58 ± 0.19and 4.70 ± 0.37 mg/g (Table 2). The BHT concentrationremaining in the LDPE layer after the coextrusion pro-cess for the 8 and 14 films were 7.88 ± 0.43 and 10.60± 0.84 mg/g, respectively. Thus, the amount of BHT lostduring processing was 1.5 and 24.3%, respectively. Ina previous work, Galindo-Arcega (2004) reported lossesof 29.7, 48.8, and 57.3% of BHT during the processingof monolayer LDPE films added with 0.5, 1, and 2 mg/g of the antioxidant, respectively. It seems that thecoextrusion reduces the loss of BHT as compared withthe extrusion of monolayer films. Moreover, the higherthe BHT added, the higher the amount lost during theextrusion process. The BHT is thermally degraded, lost


Table 2. Effect of the fabrication process on the butylated hydroxytol-uene (BHT) level in the coextruded films

BHT

In the LDPE layerIn the coextruded film

Film1 (mg/g)2 (%)3 (mg/g)2

0 0 0 08 7.88 ± 0.43 98.50 3.58 ± 0.1914 10.60 ± 0.84 75.71 4.70 ± 0.37

1Coextruded films fabricated with 0, 8, and 14 mg/g of BHT in thelow density polyethylene (LDPE) layer.

2Values are means ± standard deviation of 3 replicates.3BHT remaining in the LDPE layer after processing the films.

by reaction while performing its function as an antioxi-dant, and also evaporated to the environment becauseof its high volatility at processing temperatures. It hasbeen reported that 10% of BHT was volatilized duringa thermal gravimetric analysis of the antioxidant at157°C (Vulic et al., 2002). Temperatures up to 165°Cwere used in the LDPE extruder in the present work.The BHT may also be changed when trapping free radi-cals from LDPE chains during processing. Other au-thors reported losses of 55.5% for a LDPE film initiallyfabricated with 0.45 mg/g of BHT (Wessling et al., 1998).In the present work, increasing the BHT concentrationadded to the resin did not lead to a proportional increasein the remaining percentage of the antioxidant in thefilm.

Figure 1. Decrease of butylated hydroxytoluene (BHT) from the 8 and 14 coextruded films in contact with Asadero cheese stored at 5°Cfor 100 d. Results are means of 3 replicates. Bars indicate standard deviation.


Release Test

Figure 1 shows the loss of BHT from the 8 and 14films during the storage at 5 ± 1°C for 100 d. The diffu-sion rates for BHT were very similar in both films. Thelevel of the antioxidant decreased from 3.58 ± 0.19 and4.70 ± 0.37 to 2.45 ± 0.12 and 3.42 ± 0.06 mg/g duringthe first 3 d of storage for films 8 and 14 (31.6 and27.4% of BHT released), respectively. The levels of BHTin the films after 20 d of contact were 1.34 ± 0.06 and1.56 ± 0.15 mg/g for film 8 and 14 (62.6 and 66.9% oforiginal BHT), respectively. This behavior means thatthe antioxidant diffusion started from the first minutesof contact. From 60 to 100 d, the BHT level remainedconstant in both films at around 1 mg/g (72 and 78%of BHT released). It is important to consider that mostof the released BHT could be at the surface of the cheesewhere it is most demanded due to the effects of thelight and the low oxygen level that could diffuse throughthe multilayer film. According to the graph, no morerelease of BHT from the films would be expected be-cause it seems that the system reached equilibrium.This assumption is valid for systems in which there isno consumption of the antioxidant by radical formationas it happens in the cheese. Therefore, an increase ofthe diffusion of BHT from the films to the cheese wouldbe expected after 100 d as the oxidation reactions pro-ceed. Also, diffusion of the BHT from the surface to theinner part of the cheese could affect this equilibrium.A rapid decrease of BHT from the LDPE films into

SOTO-CANTU ET AL.16

Figure 2. Determination of D for the release of butylated hydroxytoluene (BHT) from films 8 (�, —) and 14 (�, – –) to Asadero cheeseat 5°C. Lines represent the diffusion of BHT to the cheese estimated by equation 1 during the first 20 d of contact. D values were calculatedby equation 2. Film 8 (�, —) y = 6.4735E-04x + 3.3414E-02. R2: 0.979. D: 6.242E-12 cm2/s. Film 14 (�, – –) y = 6.4807E-04x + 5.8151E-03.R2: 0.999. D: 6.256E-12 cm2/s.

different foods and food simulants has been reportedat different temperatures. Half of the BHT from a film(0.85 mg/g) in contact with oil was lost to the environ-ment in less than 6 h at 25°C (Galindo-Arcega, 2004).The loss of BHT from a packaging (LDPE) for oatmealoccurred via diffusion through the material, followedby volatilization from the film surface, being sorptiononto the oatmeal or diffusion to the surrounding atmo-sphere the final result (Wessling et al., 2000). There-fore, the rapid decrease of BHT is due to its relativelysmall size, rate of evaporation, solubility in fats, andhigh diffusion ability in the LDPE matrix.

Determination of D. Figure 2 shows the diffusiongraphs of BHT from the films to Asadero cheese consid-ering the first 20 d of contact at 5°C. Very similar Dvalues for films 8 and 14 were calculated as 6.24E-12and 6.26E-12 cm2/s, respectively. Limm and Hollifield(1996) reported a D of 8.3E-09 cm2/s for BHT in LDPEin contact with corn oil at 60°C. The thousand timeshigher value reported is explained by a high experimen-tal temperature (55°C higher than the cheese experi-ment) and also by the assumption that some compo-nents of the oil penetrated the LDPE films increasingthe BHT diffusion. More similar D values (3.7E-11 and4.2E-12 cm2/s) were reported by Till et al. (1982) formigration of BHT from a HDPE plaque (235-�m thick-ness and 100 mg of BHT/kg) to corn oil at 40 and 21°C,


respectively. In this case, the differences in tempera-tures and the higher crystallinity of HDPE contributedto the low D values. Crystallinity introduces tortuosityin the diffusion path of BHT through the HDPE poly-mer, giving similar D values when HDPE is in contactwith oil at 21°C and LDPE with cheese at 5°C. Most Dvalues for BHT migration have been calculated for filmsin contact with liquid foods or food simulants. As ex-pected, in the present work low values of D for BHTfrom the LDPE layer were obtained for coextruded filmsin contact with a solid food product under all the as-sumptions considered.

Compliance with Legislation. Assuming that allBHT released from films 8 and 14 migrated into thecheese during the 60 to 100 d of storage, concentrationsof 32.2 and 49.1 mg of BHT/kg can be estimated inthe packed cheeses, respectively. Due to the diffusionequilibrium at about 1 mg/g of BHT in the films, thesefigures are the maximum level of the antioxidant thatcould have been found in the cheese. Considering thelimit for the direct addition of BHT to foods is 200 mg/kg of fat (Code of Federal Regulations, 2007a) and thefat proportion of the Asadero cheese used in this workwas 24%; the calculated legal limit of direct addition ofBHT particularly to this cheese is 48 mg/kg. Therefore,the BHT released by film 8 complied with this legallimit but film 14 did not. Consequently, film 14 was


Figure 3. Oxidation intensity of Asadero cheese packed in the 8 (gray) and 0 (white) coextruded films and stored at 5°C for 100 d. Onthe y-axis, 0 means nonoxidized and 13 means highly oxidized. Results are means of 5 panelist responses. Bars indicate standard deviation.

discarded from the sensory analysis for safety reasons.It is important to consider that in a real case, the cheesepacked in the film 14 probably would never reach the49.1 mg/kg of BHT estimated because it might havebeen used to trap radicals from the cheese fat portion.However, the product of this reaction is still an additivein the cheese. Therefore, this type of loss was ignored tofollow a criterion to assure compliance with legislation,especially because the addition process of the antioxi-dant to the food was different from that for which thelegal limit was established. In the case of a food productin which consumer health is involved, it is better tooverestimate the BHT concentration than to underesti-mate it.

Cheese Oxidation

Preliminary studies (Soto-Cantu, 2007) found thatTHF could be used as an indicator for oxidation duringthe storage of antioxidant-packed Asadero cheese be-cause its production was inhibited by the direct additionof BHT. However, in the present experiment, THF wasonly quantified in the cheese packed in the film 0 at 60d of storage. At this time, concentrations from <12 to24.5 �g/g of THF were quantified in a triplicate (1 repli-cate below the LOQ of the analytical procedure, 2around 24.5 �g/g). Samples of cheese packed in thethree films at 20, 40, 80, and 100 d of storage showedlevels of THF below the LOQ of the analytical proce-


dure. It is important to point out that even though theTHF was found in one sampling time, it was in thecheese packed in the film with no antioxidant. In an-other work, Barbieri et al. (1994) identified 6 furans in21 samples of Parmesan cheese (24 mo old), THF beingone of the most abundant in the headspace of all thesamples. During the storage of dairy products, forma-tion and disappearance of volatile compounds is com-mon due to the oxidation reactions. That could be thereason why the THF behaved as it did in the present ex-periment.

Sensory Analysis

Oxidation odor intensity results for cheese packed infilms 0 and 8 are shown in Figure 3. Results for cheesepacked in film 14 are not presented because this treat-ment did not comply with the BHT limit stated in theregulations (Code of Federal Regulations, 2007a) andthe panelists should not have been exposed to levels ofBHT higher than 200 mg/kg of the fat portion (esti-mated as 49.1 mg/kg of cheese in this case). The cheesepacked in film 8 maintained averages of intensity ofoxidized odor from 6 to 8 until the end of storage. Al-though there were nonsignificant differences (P > 0.05),the high oxidized odor detected by the panelists showedlower standard deviations at 80 and 100 d of storagein cheese packed in the film with no antioxidant. Inother words, more panelists detected odor levels close

SOTO-CANTU ET AL.18

to the highest oxidized level at these storage times. Theoxidized odor detected at 80 and 100 d cannot be relatedto the THF found at 60 d of storage because this volatilecompound was not found at the end of storage. Thedisappearance of the THF produced could be due to itsreaction with other cheese components. Therefore, theoxidized odor detected by the panelists was due to thepresence of other volatile compounds that unfortu-nately were not quantified in the present work. TheTHF could be produced before the first 20 d of storagelike in the case of Samso cheese packed in modifiedatmosphere and stored at 5°C during 21 d (Juric et al.,2003). Future research should be directed to identifyvolatile products of Asadero cheese oxidation that couldbe correlated with the oxidized odor detected by the pan-elists.

CONCLUSIONS

In this work, an antioxidant active packaging wasdeveloped consisting of a layer made of LDPE to which8 mg/g of BHT was added and coextruded with a layerof PA6/66. This active packaging was found to maintainthe same levels of oxidation odor from 20 to 100 d ofstorage of Asadero cheese at 5°C. Most of the BHT wasdiffused from the LDPE layer to the cheese during thefirst 20 d of storage at 5°C. The release of BHT fromthe film added with 8 mg/g of the antioxidant in theLDPE layer complied with the legal limit in the cheese.However, the film added with 14 mg/g of the antioxidantin the LDPE layer could exceed that limit if all theBHT is released to the cheese. Therefore, a higher BHTconcentration film may be needed for a more effectiveoxidation odor delay from Asadero cheese, but migra-tion would exceed the legal limit of the antioxidant.

ACKNOWLEDGMENTS

This work was supported by funds from SAGARPA/CONACYT/COFUPRO (2003 C01 095), Fundacion Pro-duce Sonora and Qualyplast (Obregon, Mexico). We arevery grateful for the assistance of Jesus VillalpandoOlmos, Ximena Valenzuela, Cipactli Yuridia MezaCueto, Juan Jose Arreola Ordaz, and Humberto Gonza-lez Rıos. This work is part of the multinational projectCYTED XI.21.

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BHT Analysis in Polyethylene Film

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